EP1872120B1 - Free thyroxine and free triiodothyronine analysis by mass spectrometry - Google Patents

Free thyroxine and free triiodothyronine analysis by mass spectrometry Download PDF

Info

Publication number
EP1872120B1
EP1872120B1 EP05851243A EP05851243A EP1872120B1 EP 1872120 B1 EP1872120 B1 EP 1872120B1 EP 05851243 A EP05851243 A EP 05851243A EP 05851243 A EP05851243 A EP 05851243A EP 1872120 B1 EP1872120 B1 EP 1872120B1
Authority
EP
European Patent Office
Prior art keywords
hormone
sample
bound
mass spectrometer
hormones
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
EP05851243A
Other languages
German (de)
French (fr)
Other versions
EP1872120A4 (en
EP1872120A2 (en
Inventor
Steven J. Soldin
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Georgetown University
Original Assignee
Georgetown University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Georgetown University filed Critical Georgetown University
Publication of EP1872120A2 publication Critical patent/EP1872120A2/en
Publication of EP1872120A4 publication Critical patent/EP1872120A4/en
Application granted granted Critical
Publication of EP1872120B1 publication Critical patent/EP1872120B1/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/78—Thyroid gland hormones, e.g. T3, T4, TBH, TBG or their receptors
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/24—Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry

Definitions

  • the invention relates to methods and kits for analyzing free thyroxine (FT4) and free triiodothyronine (FT3) thyroid hormones by mass spectrometry.
  • FT4 free thyroxine
  • FT3 free triiodothyronine
  • Hormones are biological messengers. They are synthesized by specific tissues (glands) and are secreted into the blood. The blood carries them to target cells where they act to alter the activities of the target cells.
  • Hormones are chemically diverse, and are generally categorized into three main groups: (1) small molecules derived from amino acids, for example thyroxine, (2) polypeptides or proteins, for example insulin and thyroid-stimulating hormone, and (3) molecules derived from cholesterol, for example steroids.
  • thyroid hormones An important class of hormone is the thyroid hormones.
  • thyroid hormones are thyroxine (T4), free thryoxine (FT4), triiodothyronine (T3) and free triiodothyronine (FT3).
  • T4 and T3 enter cells and bind to intracellular receptors where they increase the metabolic capabilities of the cell by increasing mitochondria and mitochondrial enzymes.
  • T4 and T3 are important in regulating a number of biological processes, including growth and development, carbohydrate metabolism, oxygen consumption, protein synthesis and fetal neurodevelopment. Synthesis of all circulating T4 and a small percentage of circulating T3 occurs on thyroglobulin molecules located within the thyroid.
  • T3 The bulk of the T3 present in the blood is produced enzymatically via monodeiodination of T4 by specific intracellular deiodinases - enzymes present in the follicular cells and the cells of target tissues [1].
  • total T4 In serum drawn from healthy human subjects, total T4 is present at about 60-fold higher concentration than total T3.
  • T4 acts as a prohormone, as the reservoir for the production of T3, the active hormone.
  • the metabolic activity associated with thyroid hormone (TH) is initiated by T3 binding to specific nuclear receptors within target cells. Thyroid hormone concentrations in blood are essential tests for the assessment of thyroid function.
  • Steroids make up another important class of hormones.
  • examples of steroid hormones include estrogens, progesterone and testosterone.
  • Estrogen is the name of a group of hormones of which there are three princi one, estradiol and estriol.
  • Estrogens and progesterone cause the development of the female secondary sexual characteristics and develop and maintain the reproductive function.
  • Testosterone develops and maintains the male secondary sex characteristics, promotes growth and formation of sperm.
  • Steroids enter target cells and bind to intracellular receptors and then cause the production of mRNA coding for proteins that manifest the changes induced by steroids.
  • Table 1 lists the common hormones and the current methods for their analysis.
  • estriol is analyzed by a radioimmunoassay utilizing radiolabelled antigen (iodine 125) in competition with unlabelled estriol in the sample, for a known amount of antibody.
  • the assay is read using a gamma counter.
  • Androstenedione is analyzed using an enzyme immunoassay comprising horseradish peroxidase. Unlabeled antigen in the sample is in competition with enzyme labeled antigen for a fixed number of antibody binding sites. The assay is read using a microtitre plate enzyme immunoassay reader.
  • chemiluminescent immunoassay Several hormones are currently analyzed using a chemiluminescent immunoassay. For example, progesterone, testosterone, cortisol and T3 are analyzed using this method.
  • the assay utilizes an assay-specific antibody-coated bead.
  • the assay is read using a photon counter.
  • a method of analyzing urinary testosterone and dihydrotestosterone glucuronides using electrospray tandem mass spectrometry has been described [23].
  • the method involves a complex system employing high performance liquid chromatography (HPLC) and a three-column two-switching valve.
  • HPLC high performance liquid chromatography
  • the shortcomings include the following: (i) the hormone glucuronides were analyzed, not the hormones, (ii) the method is applicable to urine only and (iii) only two analytes were analyzed simultaneously, (iv) the limit of detection (LOD) was 200 pg ml -1 for testosterone and the limit of quantification was 10 ug L -1 for dihydrotestosterone and (v) the method is complex.
  • LOD limit of detection
  • Another publication discloses a method for the determination of estradiol in bovine plasma by an ion trap gas chromatography-tandem mass spectrometry technique [24].
  • the shortcomings include the following: (i) only one analyte was analyzed, (ii) 4 ml of plasma was required for the analysis of one analyte, (iii) the limit of detection was 5 pg ml -1 , and (iv) derivation was required because the method employs gas chromatography.
  • a gas chromatography mass spectrometry method to analyze the production rates of testosterone and dihydrosterone has been disclosed [26].
  • US 2004/0235188 describes use of mass spectrometry for the analysis of hormones, including the analysis of thyroid hormones, and total T3 and T4 in a sample.
  • the applicant's teaching provides a fast and accurate method of hormone analysis and quantification using a mass spectrometer.
  • a plurality of hormones can be analyzed simultaneously or sequentially.
  • the procedure allows for as little as 100 ⁇ L of a sample to be analyzed.
  • minimal sample preparation time is required.
  • hormone analysis permits the analysis of hormones in a number of complex matrices as they might be found in nature, e.g. the human body.
  • hormone analysis can be performed on samples of blood, saliva, serum, plasma and urine.
  • FT4 hormone a method for mass spectrometric analysis of a sample containing or suspected of containing free thyroxine (FT4) hormone, comprising the steps:
  • a system for the mass spectrometric analysis of a sample containing or suspected of containing FT4 comprising (a) reagents for separating FT4 from the sample, including internal standards, (b) reagents for analyzing FT4 hormone using a mass spectrometer, and (c) a mass spectrometer.
  • kits for use in mass spectrometric analysis of a sample containing or suspected of containing FT4 comprising (a) reagents for separating FT4 from the sample, (b) reagents for analyzing the FT4 using a mass spectrometer, (c) a solution of FT4, and (d) instructions for analyzing the FT4 using a mass spectrometer.
  • Figure 1 is a mass spectrum of a sample of plasma containing T4 and T3.
  • Figure 2 is a mass spectrum of a globulin standard containing T4 and T3.
  • Figure 3 is a typical tandem mass spectrometric chromatogram obtained for T4 and T3 for a plasma sample.
  • Figure 4 is a graph showing T3 measured by Isotope Dilution Tandem Mass Spectrometry vs. Immunoassay.
  • Figure 6 is a graph showing a typical chromatogram for free T4 (11.2 pg/mL) and deuterated internal standard.
  • Figure 7 is a graph showing the effect of temperature on FT4 by tandem mass spectrometry and ultrafiltration.
  • Figure 8 is graph showing the comparison of the tandem mass spectrometric method with the equilibrium dialysis method for the measurement of free T4.
  • Figure 9 is a graph showing the comparison of the tandem mass spectrometric method with the direct immunoassay method on the Dade RxL Dimension for the measurement of free T4.
  • Figure 10a, b, and c are a series of mass spectrums showing the analysis of FT4 (a), FT3 (b), and FT4-d2 (c) using an API 5000TM.
  • the applicant's teaching provides methods of analysis for hormones.
  • the hormones may include:
  • Any sample containing or suspected of containing a hormone can be used, including a sample of blood, plasma, serum, urine or saliva.
  • the sample may contain both free and conjugated or bound hormones.
  • a sample size of at least about 100 ⁇ L for hormones generally, or at least about 700 ⁇ L for steroid hormones when using API 3000TM, or 200 to 500 ⁇ L for steroid hormones when using the API 4000TM or API 5000TM, can be used.
  • a sample size of 500 to 600 ⁇ L for FT4 and FT3 can be used when using the API 4000TM or API 5000TM.
  • the sample may be de-proteinated. This can be done by conventional techniques known to those skilled in the art. For example, a sample can be de-proteinated with acetonitrile, containing internal standard, followed by vortexing and centrifugation.
  • the internal standard may be, for example, the deuterated hormone.
  • FT4 may be separated from other compounds, including bound T4 by centrifugation using an ultrafiltration device. After centrifugation, the ultrafiltrate will contain FT4, while the bound T4 and other compounds will be unable to pass through the filter.
  • the hormones are then introduced into a mass spectrometer.
  • the separation step and step of introducing the hormones into a mass spectrometer can be combined using a combined liquid chromatography spectrometry apparatus (LC/MS). This procedure is based on an online extraction of the injected sample with subsequent introduction into the mass spectrometer using a built-in switching valve.
  • LC/MS liquid chromatography spectrometry apparatus
  • the methods employ isotope dilution mass spectrometry.
  • the hormones are subjected to ionization.
  • Various ionization techniques can be used. For example, photoionization, electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and electron capture ionization may be used. Electrospray ionization can be utilized when analyzing thyroid hormones.
  • any tandem-mass spectrometer including hybrid quadrupole-linear ion trap mass spectrometers and liquid chromatography-tandem mass spectrometers such as the API 3000 TM mass spectrometer and the API 4000 TM mass spectrometer, described in U.S. patents 4,121,099 ; 4,137,750 ; 4,328,420 ; 4,963,736 ; 5,179,278 ; 5,248,875 ; 5,412,208 ; and 5,847,386 (Applied Biosystems/MDS SCIEX, Foster City, Calif./Concord Ontario, Canada).
  • a spectrometer with a turbo spray ion source such as the API 2000 TM and API 3000 TM mass spectrometers can be used.
  • the API 4000 TM mass spectrometer can be used.
  • the API 5000 TM mass spectrometer can be used.
  • the API 5000 TM mass spectrometer can be used.
  • Ionization may be performed by utilizing the mass spectrometer in the negative or the positive mode, depending on a particular analyte's tendency to give rise to a particular ion form, as is known to those skilled in the art.
  • the spectrometer is employed in the negative mode.
  • Hormones are identified on the basis of the mass to charge ratio of their molecular ions and fragment ions, as is known to those skilled in the art. When the hormones are purified by liquid chromatography, they can also be identified by their retention times.
  • Hormones are quantified by their intensity as determined in the mass spectrometer in counts per second. Calibration curves for known concentrations of the hormones are established for comparison.
  • Kits for use in mass spectrometric analysis of a sample comprising or suspected of comprising FT4, FT3 or both are also described.
  • the kits are assembled as is known to those skilled in the art.
  • the kits can comprise, for example, reagents for separating the hormone from the sample, reagents for analyzing the hormone using a mass spectrometer, a solution of the hormone, and instructions.
  • the eluant was introduced into an ion-spray ionization chamber and analyzed by API 2000 TM mass spectrometer using the negative mode.
  • the mass/charge ratios for T4 and T3 ions is 775.8 and 650 respectively.
  • the ionization may be by electrospray using a turboionspray chamber.
  • a sample of 100 ⁇ L of plasma was used. Proteins were precipitated with 150 ⁇ L of acetonitrile, containing an internal standard of deuterated T 4 and vortexed. The sample was centrifuged, and 200 ⁇ L of the supernatant was injected onto a C-18 column coupled to a tandem mass spectrometer (LC/MS/MS). The column was washed with 20% methanol in 5mM ammonium acetate for 3 minutes. The valve on the column was switched and the sample was eluted in a methanol gradient of 20 to 100%. The total run time was 7 minutes. Slight adjustments to the volumes, concentrations and times described can be made by those skilled in the art.
  • a sample of the eluant was introduced into an ion-spray ionization chamber and analyzed by an API 3000 TM mass spectrometer using the negative mode.
  • the ionization may be by electrospray using a turboionspray chamber.
  • Figure 1 and Figure 2 shows the mass spectrums generated for T3 and T4.
  • This example describes an isotope dilution tandem mass spectrometry method for the simultaneous determination of T4 and T3 in serum.
  • the method is accurate, specific, precise (%CVs between 3.5 and 9.0), simple - requiring no extraction and only protein precipitation, and fast. For example it can be done in less than seven minutes.
  • T3, T4 and internal standard (IS) were prepared separately to obtain a concentration of 1mg/mL for each.
  • 40% ammonium hydroxide (v/v) in methanol was used as a solvent.
  • the analyte stock solutions were diluted with methanol to obtain the spiking solutions.
  • the solutions were stored at 4°C and could be used for several months.
  • Standards for the calibration curve in the range of 0.325 to 5 ng/mL for T3 and 12.5 to 200 ng/mL for T4 were prepared by adding the analyses to 3 % human ⁇ -globulin (volume of spiking solution ⁇ 2% of final volume).
  • Quality control (QC) samples (Diagnostic Product Corp., Los Angeles, USA) at low, medium and high levels were used.
  • a solution of 50-ng/mL d 2 -T4 in methanol was used as the internal standard.
  • Serum or plasma samples were thawed at room temperature. 150 ⁇ L of IS solution was added to aliquots of 100 ⁇ L of the serum or plasma sample. After 30 seconds of vortex mixing, the samples were stored for 10 minutes at room temperature to allow complete protein precipitation. The samples were centrifuged for 10 minutes at 15,000 rpm and 100 ⁇ l of supernatant was injected into the LC-MS-MS system.
  • the procedure used is based on an online extraction/cleaning of the injected samples with subsequent introduction into the mass-spectrometer by using a built-in Valco switching valve.
  • the switching valve was activated, the column was flushed with water/ methanol gradient at flow rate 0.5 mL/min and the samples were introduced into the mass-spectrometer.
  • the gradient parameters used are shown in Table 4.
  • T4 was measured by the Dade RxL DimensionTM (Dade-Behring Diagnostics, Glasgow, DE) and T3 by the DPC ImmuliteTM (Diagnostic Product Corporation, Los Angeles, CA) according to the manufacturer's specifications.
  • Figure 3 shows a typical tandem mass spectrometric chromatogram obtained for T3 and T4 (T4 m/z (776/127); D 2 T4 m/z (778/127); T3 m/z (650/127)).
  • the lower limit of quantitation of the mass spectrometry method was found to be 0.15 ng/mL for both T3 and T4. Detection limit was around 0.062 ng/mL.
  • Table 2 clearly indicates that current IAs for T4 and T3 lack specificity and give mean results differing by a factor of approximately 2 in the College of American Pathologists Proficiency Testing (CAP PT) programs.
  • Total hormone assays necessitate the inclusion of a displacing agent (such as salicylate) to release the hormone from its binding proteins [28].
  • a displacing agent such as salicylate
  • T3 is ten-fold lower in concentration compared with T4 in blood it therefore presents both a technical sensitivity and precision challenge despite the use of a higher specimen volume.
  • a reliable high-range T3 measurement is critical for diagnosing hyperthyroidism
  • a reliable normal-range measurement is also important for adjusting antithyroid drug dosage and detecting hyperthyroidism in sick hospitalized patients, in whom a paradoxically normal T3 value may indicate hyperthyroidism.
  • T4 comparisons (0.931) is significantly better than for the T3 comparisons (0.848) ( Figures 4 and 5 ).
  • T3 by tandem mass spectrometry gave slightly higher results than those obtained by the DPC ImmuliteTM ( Figure 4 ). While this is true for children, preliminary data for non-pregnant and pregnant women indicates a very poor correlation for T3 in both groups (r between 0.407-0.574) (i.e. there is a poor correlation between DPC Immulite and the method of the present teaching in both non-pregnant and pregnant women).
  • the isotope dilution tandem mass spectrometric method of the applicant's teaching is rapid (less than 7 minutes), accurate (provides the true result as has been assessed by recovery studies), specific (measures only the analyte it purports to measure), precise (low %CV) and easy to perform.
  • Thyroxine was purchased from Sigma (St Louis, MO). A stable deuterium-labeled internal standard, L-thyroxin-d 2 was synthesized according to procedures described in the literature (29, 30) by Dr Tomas Class from the Chemistry Department at Georgetown University. HPLC grade methanol was purchased from VWR Scientific. All other chemicals were of analytical grade and were purchased from Sigma.
  • T4 and internal standard were prepared separately to obtain concentration of 10 mg/mL for each using 40% ammonium hydroxide (v/v) in methanol as a solvent.
  • the analyte stock solutions were diluted with methanol to obtain the spiking solutions.
  • the solutions were stored at -20°C and could be used for several months.
  • Standards for the T4 calibration curve in the range of 2.5-50 pg/mL were prepared by adding the analytes to water.
  • a solution of 0.05 ng/mL d 2 -T4 in methanol was used as internal standard.
  • Serum or plasma samples were obtained from greater than 42 healthy pregnant and 29 non-pregnant women in a study approved by the Institutional Review Board (IRB) and were thawed at room temperature.
  • 0.6 ml samples were filtered through Centrifree YM-30 ultrafiltration devices (30,000 MW cut-off, Millipore, Bedford, MA) by centrifugation employing the Eppendorf temperature controlled centrifuge (model # 5702 R, Eppendorf, AG, Hamburg) and using a fixed angle rotor at 2900 rpm and a temperature of 25° for 1 hour.
  • 180 ⁇ L IS [0.05ng/mL] was added to 360 ⁇ L ultrafiltrate and 400 ⁇ L was injected onto the C-18 column of the LC/MS/MS system.
  • This ultrafiltration process replaces the dialysis step of the classic equilibrium dialysis method.
  • the ultrafiltration step includes removal of all proteins having a molecular weight of greater than 30,000.
  • the liquid chromatography step can be used to further separate and pur
  • the procedure used is based on an online extraction/cleaning of the injected samples with subsequent introduction into the mass-spectrometer by using a built-in Valco switching valve.
  • 400 ⁇ L of the sample was injected onto the Supelco LC-18-DB (3.3 mm x 3.0 mm, 3.0 ⁇ m ID) chromatographic column equipped with a Supelco Discovery C-18 (3.0 mm) guard column, where it underwent cleaning with 20% (v/v) methanol in 5 mM ammonium acetate pH-4.0 at flow rate 0.8 mL/min.
  • the switching valve was activated, the column was flushed with a water/methanol gradient at flow rate of 0.6 mL/min and the samples were introduced into the mass-spectrometer.
  • the gradient parameters that were used are shown in Table 10.
  • the free T4 chromatogram is shown in Figure 6 .
  • Tables 9 and 10 provide the analytical parameters employed for the tandem mass spectrometric method.
  • Figure 6 shows a typical chromatogram for free T4 measured by tandem mass spectrometry using the method described. The time per analysis is approximately 8.5 minutes although a steeper gradient could shorten this to about 6 minutes.
  • the Eppendorf centrifuge allows for the centrifugation of 30 tubes simultaneously so that the total run time for 30 patient samples at the 25°C temperature used is 1 hour plus 3 hours and 15 minutes, or 4 hours and 15 minutes.
  • This ultrafiltration plus LGMS/MS assay is considerably quicker than the time consuming equilibrium dialysis method. The latter requires 16-18 hour dialysis at 37°C followed by an immunoassay and therefore the turn-around-time is several days.
  • a sample of 500 to 1000 ⁇ L of plasma is used. Proteins are precipitated with 150 ⁇ L of acetonitrile and vortexed. The sample is centrifuged, and 200 ⁇ L of the supernatant is injected onto a C-18 column coupled to a tandem mass spectrometer (LC/MS/MS). The column is washed with 20% methanol in 5mM ammonium acetate for 3 minutes. The valve on the column is switched and the sample is eluted in a methanol gradient of 20 to 100%. The total run time is 10 minutes. Slight adjustments to the volumes, concentrations and times described can be made, as is known to those skilled in the art.
  • a sample of the eluant is introduced into an ion-spray ionization chamber and analyzed by API 3000TM mass spectrometer using the negative mode for thyroid hormones in the sample.
  • Steroid hormones in the sample are ionized by photoionization, with the spectrometer in the negative or positive mode.
  • Analysis in the positive mode is typically made for DHEA, Aldosterone, Cortisol, 11-Deoxycortisol, Androstenedione, Testosterone, Estradiol, 17-OH Progesterone, Progesterone, Allopregnalone, Vitamin D, 25,hydroxyl Vitamin D, 1,25 dihydroxy Vitamin D, corticosterone and aldosterone, whereas analysis in the negative mode is typically made for 16-OH Estrone, 2-OH Estrone, Estriol and DHEAS. However, it is possible to analyze any of the hormones in either positive or negative mode.
  • FT3 was analyzed by the same method as FT4 (Example 4), except for the analysis of the same transition ions for total T3 and using the API 5000TM mass spectrometer.
  • FT4 and FT3 concentrations Patients with either hyperthyroidism or hypothyroidism require frequent assessment of thyroid function through measurement of their FT4 and FT3 concentrations. Further, people with thyroid ablation require thyroid replacement therapy, such as synthroid. Measurement of their FT4 and FT3 concentrations is important when assessing their dosage regimen. Accordingly, an efficient assay method for the simultaneous analysis of FT3 and FT4 is beneficial.
  • FT4 and FT3 were analyzed simultaneously by a similar method of Example 4 except using the API 5000TM mass spectrometer.
  • 100 ⁇ L mixture of T3 (25pg/mL) and T4 (1 ng/mL) with internal standard T4-d2 were injected onto the column by autosampler, and the column was washed by 20% MeOH buffer for 2 minutes. Gradient elution started from 20% MeOH to 100% MeOH in 2 minutes after the Valco valve was activated at 2 minutes, and then kept at 100% for another 2 minutes. The retention times were: T3, 4.34 minutes, T4, 4.60 minutes, and T4-d2, 4.61 minutes.
  • Figure 10 shows the mass spectrums of the analytes. Standard curves for FT3 (1-25 pg/ml) and FT4 (5-50 pg/ml) can be run with the analysis of the samples.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Endocrinology (AREA)
  • Pathology (AREA)
  • Microbiology (AREA)
  • General Physics & Mathematics (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Food Science & Technology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Description

  • The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way.
    • FIELD
  • The invention relates to methods and kits for analyzing free thyroxine (FT4) and free triiodothyronine (FT3) thyroid hormones by mass spectrometry.
    • BACKGROUND
  • Hormones are biological messengers. They are synthesized by specific tissues (glands) and are secreted into the blood. The blood carries them to target cells where they act to alter the activities of the target cells.
  • Hormones are chemically diverse, and are generally categorized into three main groups: (1) small molecules derived from amino acids, for example thyroxine, (2) polypeptides or proteins, for example insulin and thyroid-stimulating hormone, and (3) molecules derived from cholesterol, for example steroids.
  • An important class of hormone is the thyroid hormones. Examples of thyroid hormones are thyroxine (T4), free thryoxine (FT4), triiodothyronine (T3) and free triiodothyronine (FT3). T4 and T3 enter cells and bind to intracellular receptors where they increase the metabolic capabilities of the cell by increasing mitochondria and mitochondrial enzymes. T4 and T3 are important in regulating a number of biological processes, including growth and development, carbohydrate metabolism, oxygen consumption, protein synthesis and fetal neurodevelopment. Synthesis of all circulating T4 and a small percentage of circulating T3 occurs on thyroglobulin molecules located within the thyroid. The bulk of the T3 present in the blood is produced enzymatically via monodeiodination of T4 by specific intracellular deiodinases - enzymes present in the follicular cells and the cells of target tissues [1]. In serum drawn from healthy human subjects, total T4 is present at about 60-fold higher concentration than total T3. T4 acts as a prohormone, as the reservoir for the production of T3, the active hormone. The metabolic activity associated with thyroid hormone (TH) is initiated by T3 binding to specific nuclear receptors within target cells. Thyroid hormone concentrations in blood are essential tests for the assessment of thyroid function.
  • Steroids make up another important class of hormones. Examples of steroid hormones include estrogens, progesterone and testosterone. Estrogen is the name of a group of hormones of which there are three princi one, estradiol and estriol. Estrogens and progesterone cause the development of the female secondary sexual characteristics and develop and maintain the reproductive function. Testosterone develops and maintains the male secondary sex characteristics, promotes growth and formation of sperm. Steroids enter target cells and bind to intracellular receptors and then cause the production of mRNA coding for proteins that manifest the changes induced by steroids.
  • The accurate analysis and quantification of hormones is becoming more important. For example, estrogen and estrogen-like compounds are playing an ever-increasing role in today's society through hormone replacement therapy. Also, the analysis and quantification of estrogen and estrogen-like compounds helps in the management of estrogen-related diseases, like breast cancer. In addition, the accurate analysis and quantification of T4 and T3 is an issue recognized by those skilled in the art. The presence of circulating iodothyronine-binding autoantibodies that interfere with total T4 and T3 immunoassays ("IAs") is a known phenomenon [2], [3], [4]. These autoantibodies may give falsely high, or falsely low values of thyroid hormone measurements depending on the assay separation method used, and are often in discordance with the clinical features [2], [3], [4]. Serum free T4 and T3 (FT4 and FT3) measurements are a way to compensate for such abnormal binding. However, technically, it is difficult to measure the free hormone concentrations since these are so low. It is easier to measure the total (free and protein-bound) thyroid hormone concentrations; total hormone concentrations are measured at nanomolar levels whereas free hormone concentrations are measured in the picomole range and to be valid, must be free from interference by the much higher total hormone concentrations.
  • Presently, the common methods of hormone analysis use immunoassay techniques. Table 1 lists the common hormones and the current methods for their analysis.
  • For example, estriol is analyzed by a radioimmunoassay utilizing radiolabelled antigen (iodine 125) in competition with unlabelled estriol in the sample, for a known amount of antibody. The assay is read using a gamma counter.
  • Androstenedione is analyzed using an enzyme immunoassay comprising horseradish peroxidase. Unlabeled antigen in the sample is in competition with enzyme labeled antigen for a fixed number of antibody binding sites. The assay is read using a microtitre plate enzyme immunoassay reader.
  • Several hormones are currently analyzed using a chemiluminescent immunoassay. For example, progesterone, testosterone, cortisol and T3 are analyzed using this method. The assay utilizes an assay-specific antibody-coated bead. The assay is read using a photon counter.
  • However, the current immunoassays are disadvantageous for the following reasons:
    1. (1) Immunoassays are specific to one hormone, therefore every hormone must be analyzed separately.
    2. (2) Numerous kits must be purchased and procedures must be learned for each hormone being analyzed.
    3. (3) Various instruments to read the results from the immunoassays must be purchased. For example, the analysis of estriol and progesterone from a sample requires both a gamma counter and a photon counter.
    4. (4) The kits for the assays can be expensive.
    5. (5) The current immunoassays lack specificity and may show approximately 15 fold difference in results using kits from different manufacturers [5].
    6. (6) The procedures involve many steps and can take a significant amount of time.
    7. (7) In the case of a radioimmunoassay, precautions are necessary because of the radioisotopes involved.
  • Immunoassays are notoriously unreliable with more and more literature published supporting their lack of specificity [6-13]. Table 2 shows the major differences reported by the College of American Pathologists program for proficiency testing of thyroid hormones that clearly illustrates the difference in specificity of the various antibodies used. For example, Table 2 shows mean results between different methods reported in the College of American Pathologists Proficiency Testing (CAP PT) Program can vary by a factor of approximately 2. Some factors such as pregnancy, estrogen therapy or genetic abnormalities in protein binding have also reportedly made immunoassay methods for T4 and T3 diagnostically unreliable [2], [3], [14], [15]. Currently serum total T4 (TT4), free T4 (FT4) and free T3 (FT3) concentrations are most commonly measured by immunoassay methods. Recently some reports of quantitative measurement of T4 and T3 by high performance liquid chromatography (HPLC), gas chromatography mass spectrometry (GC-MS), liquid chromatography mass spectrometry (LC-MS) or tandem mass spectrometry (LC-MS/MS) were published [16-20]. All those methods required extraction, derivatization and even prior chromatographic separation that are very time-consuming [21], [22].
  • More recently, hormones have been analyzed and quantified by mass spectrometry. However, there are several disadvantages to these methods.
  • For example, a method of analyzing urinary testosterone and dihydrotestosterone glucuronides using electrospray tandem mass spectrometry has been described [23]. The method involves a complex system employing high performance liquid chromatography (HPLC) and a three-column two-switching valve. The shortcomings include the following: (i) the hormone glucuronides were analyzed, not the hormones, (ii) the method is applicable to urine only and (iii) only two analytes were analyzed simultaneously, (iv) the limit of detection (LOD) was 200 pg ml-1 for testosterone and the limit of quantification was 10 ug L-1 for dihydrotestosterone and (v) the method is complex.
  • Another publication discloses a method for the determination of estradiol in bovine plasma by an ion trap gas chromatography-tandem mass spectrometry technique [24]. The shortcomings include the following: (i) only one analyte was analyzed, (ii) 4 ml of plasma was required for the analysis of one analyte, (iii) the limit of detection was 5 pg ml-1, and (iv) derivation was required because the method employs gas chromatography.
  • A method for analysis of 17-hydroxyprogesterone by HPLC electrospray ionization tandem mass spectrometry from dried blood spots has also been described [25]. However, this method analyses only one analyte at a time, and requires liquid-liquid extraction, which is laborious and time consuming, with sample extraction alone taking 50 minutes to complete.
  • A gas chromatography mass spectrometry method to analyze the production rates of testosterone and dihydrosterone has been disclosed [26].
    US 2004/0235188 describes use of mass spectrometry for the analysis of hormones, including the analysis of thyroid hormones, and total T3 and T4 in a sample.
  • Finally, there is no known method of analyzing free thyroxine (FT4) or free triiodothyronine (FT3) by mass spectrometry. Most laboratories perform FT4 testing routinely employing the analogue (direct) immunoassay approach on one of the major clinical chemistry platforms. This approach is not universally accepted and has been the subject of criticism (29). There are frequent occasions when the validity of the FT4 result generated in this manner is questioned. For this reason a "reflex" testing for all direct FT4's <2.5th percentile is often done to diagnose hypothyroidism. These are sent out for FT4 measurements employing the current gold standard of equilibrium dialysis. This is also done for samples when the direct FT4 is >97.5th percentile and the TSH is normal. Approximately 50% of these FT4 send-outs have results within the normal range when measured by equilibrium dialysis and are therefore false positives by the direct FT4 method. However, the equilibrium dialysis procedures are time-consuming and expensive. Similarly, FT3 is also currently measured by immunoassay.
    US - 6153440 describes methods for the simultaneous measurement of T3 and T4 in a sample, using direct equilibrium dialysis to separate free T3 and free T4 from bound T3 and bound T4, followed by an immunoassay. TABLE 1: METHODS AND INSTRUMENTS FOR STEROID AND THYROID HORMONES [1]
    Analyte Percentage of Use Instrument Method
    Androstenedione
    35% DSL solid EIA
    11-Deoxycortisol 50% ICN Immuchem DA RIA
    DHEA Sulfate 39% DPC Immulite ECIA
    Estradiol 16% Bayer ADVIA Centaur FIA
    Estriol, unconjugated 25% DSL liquid RIA
    Estriol, Total 50% DPC Coat-a-Count RIA
    17-Hydroxyprogesterone 51% DPC Coat-a-Count RIA
    Progesterone 23% Bayer ADVIA Centaur CIA
    Testosterone 29% Bayer ADVIA Centaur CIA
    Testosterone, Free 65% DPC Coat-a-Count RIA
    Aldosterone 76% DPC Coat-a-Count RIA
    Cortisol
    25% Bayer ADVIA Centaur CIA
    Corticosterone
    T3 29% Abbott Axsym FPIA
    T3, Free 31% Bayer ADVIA Centaur CIA
    T4
    30% Abbott Axsym FPIA
    T4, Free 34% Abbott Axsym FPIA
    RIA: Radioimmunoassay
    EIA: Enzyme Linked Immunoassay
    FPIA: Fluorescence Polarization Immunoassay
    Table 2: Problems with Immunoassays: Data acquired from CAP PT Program 2003
    Analyte Mean CAP Result for Method Giving Lowest Value Mean CAP Result for Method Giving Highest Value
    Triiodothyronine (ng/dL) 108.5 190.2
    364.8 610.1
    Thyroxine (ug/dL) 5.64 10.09
    1.64 3.65
    8.73 13.12
    Table 2: Problems with Immunoassays: Data acquired for samples from the CAP PT Program 2003.
    • SUMMARY
  • The applicant's teaching provides a fast and accurate method of hormone analysis and quantification using a mass spectrometer.
  • A plurality of hormones can be analyzed simultaneously or sequentially. The procedure allows for as little as 100 µL of a sample to be analyzed. In addition, minimal sample preparation time is required.
  • The applicant's teaching permits the analysis of hormones in a number of complex matrices as they might be found in nature, e.g. the human body. For, example, hormone analysis can be performed on samples of blood, saliva, serum, plasma and urine.
  • There are several features to this teaching:
    1. (1) It provides a total and specific analysis for hormones in a sample. The present method allows for the analysis of many hormones simultaneously or sequentially.
    2. (2) The procedure does not require an immunoprecipitation reaction. The majority of other methods for hormone analysis required an immunoassay. Immunoassays are expensive, specific to a particular analyte and involve several steps.
    3. (3) The present teaching requires minimal sample preparation time. For example, preparing a sample for hormone analysis can be done within 6 minutes.
    4. (4) The procedure does not require a large sample size. A plasma or serum sample can be as small as 100 µL for thyroid hormones. For FT4 and FT3 the sample can be between 500 and 600 µL. The current methods for hormone analysis that utilize mass spectrometry require 4-15 mL of plasma.
    5. (5) The methods use simple preparation techniques that are easy to use and highly reproducible.
    6. (6) The methods permit analysis to be performed on a variety of sample types.
    7. (7) The methods permit the analysis of hormones in a sample of saliva or urine which permits simple sample acquisition and the remote submission of samples to a clinic for analysis. In previous other clinical methods, samples are taken by invasive means directly from the patient in a clinic.
    8. (8) The analysis by mass spectrometry is highly accurate. In addition, the procedure of the present methods are highly reproducible.
    9. (9) The methods permit the analysis of a wide range of hormone concentrations. In addition, the limit of detection can be fairly low.
  • Accordingly, there is provided a method for mass spectrometric analysis of a sample containing or suspected of containing free thyroxine (FT4) hormone, comprising the steps:
    1. (a) providing a sample containing or suspected of containing FT4 hormone;
    2. (b) separating FT4 hormone from the sample and from bound T4 by centrifugation using an ultrafiltration device;
    3. (c) collecting FT4 hormone separated from bound T4; and
    4. (d) analyzing FT4 hormone separated from bound T4 using a mass spectrometer.
    The method may additionally comprise analysis of free triiodothyronine (FT3) hormone, the method comprising the steps of:
    1. (a) providing the sample containing or suspected of containing FT4 and FT3;
    2. (b) separating FT4 hormone and FT3 hormone from the sample and from bound T4 and bound T3;
    3. (c) collecting FT4 and FT3 hormone separated from bound T4 and bound T3; and
    4. (d) analyzing FT4 and FT3 hormone separated from bound T4 and bound T3 using a mass spectrometer.
  • We also describe a system for the mass spectrometric analysis of a sample containing or suspected of containing FT4, comprising (a) reagents for separating FT4 from the sample, including internal standards, (b) reagents for analyzing FT4 hormone using a mass spectrometer, and (c) a mass spectrometer.
  • We also describe a kit for use in mass spectrometric analysis of a sample containing or suspected of containing FT4 comprising (a) reagents for separating FT4 from the sample, (b) reagents for analyzing the FT4 using a mass spectrometer, (c) a solution of FT4, and (d) instructions for analyzing the FT4 using a mass spectrometer.
  • We also describe use of a mass spectrometer for analyzing a sample containing or suspected of containing FT4, FT3 or both.
  • These and other features of the applicant's teachings are set forth herein.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • The skilled person in the art will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the applicant's teachings in any way.
  • The methods, including the best approaches known to the inventors, can be better understood with reference to the following description taken in combination with the following drawings, in which:
  • Figure 1 is a mass spectrum of a sample of plasma containing T4 and T3.
  • Figure 2 is a mass spectrum of a globulin standard containing T4 and T3.
  • Figure 3 is a typical tandem mass spectrometric chromatogram obtained for T4 and T3 for a plasma sample. T4 m/z (776/127); D2T4 m/z (778/127); T3 m/z (650/127).
  • Figure 4 is a graph showing T3 measured by Isotope Dilution Tandem Mass Spectrometry vs. Immunoassay. IA=075 MS + 0.21; r=0.848; Sy,x=0.1956; n=49.
  • Figure 5 is a graph showing T4 measured by Isotope Dilution Tandem Mass Spectrometry vs. Immunoassay. IA=1.13 MS-8.99; r=0.931; Sy,x=9.54; n=50.
  • Figure 6 is a graph showing a typical chromatogram for free T4 (11.2 pg/mL) and deuterated internal standard.
  • Figure 7 is a graph showing the effect of temperature on FT4 by tandem mass spectrometry and ultrafiltration.
  • Figure 8 is graph showing the comparison of the tandem mass spectrometric method with the equilibrium dialysis method for the measurement of free T4.
  • Figure 9 is a graph showing the comparison of the tandem mass spectrometric method with the direct immunoassay method on the Dade RxL Dimension for the measurement of free T4.
  • Figure 10a, b, and c are a series of mass spectrums showing the analysis of FT4 (a), FT3 (b), and FT4-d2 (c) using an API 5000™.
    • DESCRIPTION OF VARIOUS EMBODIMENTS
  • The applicant's teaching provides methods of analysis for hormones. The hormones may include:
    • Dehydroepiandrosterone (DHEA)
    • Dehydroepiandrosterone sulphate (DHEAS)
    • Aldosterone
    • Cortisol
    • Corticosterone
    • 11-Deoxycortisol
    • Androstenedione
    • Testosterone
    • Estradiol
    • 17-OH Progesterone
    • Progesterone
    • Allopregnanolone
    • 16-OH Estrone
    • 2-OH Estrone
    • Estrone
    • Estriol
    • Vitamin D, and its metabolites 25hydroxyvitamin D and 1,25 dihydroxyvitamin D. thyroxine
    • free thyroxine
    • triiodothyronine
    • free triiodothyronine
    • catecholamines
    • metanephrines
    • other steroid hormones
    • other thyroid hormones
    • other small peptide hormones
    • other amines
  • Any sample containing or suspected of containing a hormone can be used, including a sample of blood, plasma, serum, urine or saliva. The sample may contain both free and conjugated or bound hormones. A sample size of at least about 100 µL for hormones generally, or at least about 700 µL for steroid hormones when using API 3000™, or 200 to 500 µL for steroid hormones when using the API 4000™ or API 5000™, can be used. A sample size of 500 to 600 µL for FT4 and FT3 can be used when using the API 4000™ or API 5000™.
    • Deproteinization
  • The sample may be de-proteinated. This can be done by conventional techniques known to those skilled in the art. For example, a sample can be de-proteinated with acetonitrile, containing internal standard, followed by vortexing and centrifugation. The internal standard may be, for example, the deuterated hormone.
    • Separation of hormones from the sample
  • The hormones are separated by centrifugation. For example, FT4 may be separated from other compounds, including bound T4 by centrifugation using an ultrafiltration device. After centrifugation, the ultrafiltrate will contain FT4, while the bound T4 and other compounds will be unable to pass through the filter.
    • Introduction of hormones into a mass spectrometer
  • The hormones are then introduced into a mass spectrometer. Optionally, the separation step and step of introducing the hormones into a mass spectrometer can be combined using a combined liquid chromatography spectrometry apparatus (LC/MS). This procedure is based on an online extraction of the injected sample with subsequent introduction into the mass spectrometer using a built-in switching valve.
    • Isotope Dilution Tandem Mass Spectrometry
  • The methods employ isotope dilution mass spectrometry.
    • Instrumentation and ionization techniques
  • The hormones are subjected to ionization. Various ionization techniques can be used. For example, photoionization, electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and electron capture ionization may be used. Electrospray ionization can be utilized when analyzing thyroid hormones.
  • The following mass spectrometers can be used: any tandem-mass spectrometer, including hybrid quadrupole-linear ion trap mass spectrometers and liquid chromatography-tandem mass spectrometers such as the API 3000™ mass spectrometer and the API 4000™ mass spectrometer, described in U.S. patents 4,121,099 ; 4,137,750 ; 4,328,420 ; 4,963,736 ; 5,179,278 ; 5,248,875 ; 5,412,208 ; and 5,847,386 (Applied Biosystems/MDS SCIEX, Foster City, Calif./Concord Ontario, Canada). When analyzing thyroid hormones, a spectrometer with a turbo spray ion source, such as the API 2000™ and API 3000™ mass spectrometers can be used. When analyzing FT4, the API 4000™ mass spectrometer can be used. When analyzing FT3, the API 5000™ mass spectrometer can be used. When analyzing FT3 and FT4 simultaneously the API 5000™ mass spectrometer can be used.
  • Ionization may be performed by utilizing the mass spectrometer in the negative or the positive mode, depending on a particular analyte's tendency to give rise to a particular ion form, as is known to those skilled in the art. Typically, for thyroid hormones, the spectrometer is employed in the negative mode.
  • Hormones are identified on the basis of the mass to charge ratio of their molecular ions and fragment ions, as is known to those skilled in the art. When the hormones are purified by liquid chromatography, they can also be identified by their retention times.
  • Hormones are quantified by their intensity as determined in the mass spectrometer in counts per second. Calibration curves for known concentrations of the hormones are established for comparison.
    • Kits
  • Kits for use in mass spectrometric analysis of a sample comprising or suspected of comprising FT4, FT3 or both are also described. The kits are assembled as is known to those skilled in the art. The kits can comprise, for example, reagents for separating the hormone from the sample, reagents for analyzing the hormone using a mass spectrometer, a solution of the hormone, and instructions.
    • EXAMPLES
  • Aspects of the applicant's teachings may be further understood in light of the following examples, which should not be construed as limiting the scope of the present teachings in any way.
    • 1. Analysis of a sample for thyroid hormones
  • A sample of 100 µL of plasma was used. Proteins were precipitated with 150 µL of acetonitrile, capped and vortexed. The sample was then centrifuged, and 200 µL of the supernatant was injected onto a Supelco LC-18-DB™ chromatographic column equipped with Supelco Discovery C-18™ guard column, coupled to a tandem mass spectrometer (LC/MS/MS). The column was washed with 20% methanol in 5mM ammonium acetate for 3 minutes. The valve was switched and the sample was eluted in 75% to 95% methanol. The total run time was 6 minutes. Slight adjustments to the volumes, concentrations and times described can be made, as is known to those skilled in the art.
  • The eluant was introduced into an ion-spray ionization chamber and analyzed by API 2000™ mass spectrometer using the negative mode. The mass/charge ratios for T4 and T3 ions is 775.8 and 650 respectively. The ionization may be by electrospray using a turboionspray chamber.
  • This demonstrates a simple method of preparing a complex biological matrix for analysis of hormone content, and a sensitive analytical method that permits the simultaneous analysis of two hormones, T3 and T4.
    • 2. Analysis of thyroid hormones using a methanol gradient to elute the hormones
  • A sample of 100 µL of plasma was used. Proteins were precipitated with 150 µL of acetonitrile, containing an internal standard of deuterated T4 and vortexed. The sample was centrifuged, and 200 µL of the supernatant was injected onto a C-18 column coupled to a tandem mass spectrometer (LC/MS/MS). The column was washed with 20% methanol in 5mM ammonium acetate for 3 minutes. The valve on the column was switched and the sample was eluted in a methanol gradient of 20 to 100%. The total run time was 7 minutes. Slight adjustments to the volumes, concentrations and times described can be made by those skilled in the art.
  • A sample of the eluant was introduced into an ion-spray ionization chamber and analyzed by an API 3000™ mass spectrometer using the negative mode. The ionization may be by electrospray using a turboionspray chamber. Figure 1 and Figure 2 shows the mass spectrums generated for T3 and T4.
  • This demonstrates a simple method of preparing a complex biological matrix for analysis of thyroid hormone content, and a sensitive analytical method that permits the simultaneous analysis of multiple hormones.
    • 3. Analysis of thyroid hormones using isotope dilution tandem mass spectrometry
  • This example describes an isotope dilution tandem mass spectrometry method for the simultaneous determination of T4 and T3 in serum. The method is accurate, specific, precise (%CVs between 3.5 and 9.0), simple - requiring no extraction and only protein precipitation, and fast. For example it can be done in less than seven minutes.
    • Chemicals and reagents
  • Standards of T4 and T3 were purchased from Sigma (St. Louis, MO, USA). A stable deuterium-labeled internal standard, L-thyroxin-d2 was synthesized according to procedures described in the literature [16], [17] by Dr Tomas Class from the Chemistry Department at Georgetown University. HPLC grade methanol was purchased from VWR Scientific. All other chemicals were of analytical grade and purchased from Sigma.
    • Solutions and standards
  • Stock solutions of T3, T4 and internal standard (IS) were prepared separately to obtain a concentration of 1mg/mL for each. 40% ammonium hydroxide (v/v) in methanol was used as a solvent. The analyte stock solutions were diluted with methanol to obtain the spiking solutions. The solutions were stored at 4°C and could be used for several months. Standards for the calibration curve in the range of 0.325 to 5 ng/mL for T3 and 12.5 to 200 ng/mL for T4 were prepared by adding the analyses to 3 % human γ-globulin (volume of spiking solution < 2% of final volume). Quality control (QC) samples (Diagnostic Product Corp., Los Angeles, USA) at low, medium and high levels were used. A solution of 50-ng/mL d2-T4 in methanol was used as the internal standard.
    • Sample preparation
  • Serum or plasma samples were thawed at room temperature. 150 µL of IS solution was added to aliquots of 100 µL of the serum or plasma sample. After 30 seconds of vortex mixing, the samples were stored for 10 minutes at room temperature to allow complete protein precipitation. The samples were centrifuged for 10 minutes at 15,000 rpm and 100 µl of supernatant was injected into the LC-MS-MS system.
    • LC/MS/MS conditions
  • An API 3000™ tandem mass-spectrometer (SCIEX, Toronto, Canada) equipped with TurboIonSpray and Shimadzu HPLC system was used to perform the analysis. Negative ion multiple reaction-monitoring (MRM) mode was used. The transitions to monitor were selected at m/z 650→ 127 for T3, m/z 776→ 127 for T4, m/z 778→ 127 for d2-T4. Nitrogen served as auxiliary, curtain and collision gas. Gas flow rates, source temperature, Ion Spray voltages and collision energies were optimized for every compound by infusion of 1µg/mL of the standard solutions in methanol at 20 µL/min and by flow-injection analysis (FIA) at LC flow rate. The main working parameters for the mass spectrometer are summarized in Table 3. Data processing was performed on Analyst 1.2 software package.
    • LC-MS-MS procedure
  • The procedure used is based on an online extraction/cleaning of the injected samples with subsequent introduction into the mass-spectrometer by using a built-in Valco switching valve. 100 µl of the sample was injected onto a Supelco LC-18-DB (3.3 cm x 3.0 mm, 3.0 µm ID) chromatographic column equipped with a Supelco Discovery C-18 (3.0 mm) Guard column, where it underwent cleaning with 20% (v/v) methanol in 5 mM ammonium acetate pH=4.0 at flow rate 0.8 mL/minute. After 3.5 minutes of cleaning the switching valve was activated, the column was flushed with water/ methanol gradient at flow rate 0.5 mL/min and the samples were introduced into the mass-spectrometer. The gradient parameters used are shown in Table 4.
    • Immunoassays for T4 and T3
  • T4 was measured by the Dade RxL Dimension™ (Dade-Behring Diagnostics, Glasgow, DE) and T3 by the DPC Immulite™ (Diagnostic Product Corporation, Los Angeles, CA) according to the manufacturer's specifications.
    • Results
  • The mass spectrometer working parameters used are shown in Tables 3 and 4.
  • Replicate sera were assayed both within-day and between-day at several concentrations. The within-day and between-day precision data is provided in Tables 5 and 6.
  • Recovery studies for T4 and T3 are shown in Tables 7 and 8. All results shown are the means of 8 replicates.
  • Figure 3 shows a typical tandem mass spectrometric chromatogram obtained for T3 and T4 (T4 m/z (776/127); D2T4 m/z (778/127); T3 m/z (650/127)).
  • Specimens were tested for T3 and T4 by both immunoassay (T3 DPC Immulite, T4 Dade Behring Dimension™ RxL) and by tandem mass spectrometry. Linear regression correlations (Prism) are shown in Figures 4 and 5.
  • The lower limit of quantitation of the mass spectrometry method was found to be 0.15 ng/mL for both T3 and T4. Detection limit was around 0.062 ng/mL.
    • Discussion
  • Evidence initially gleaned from both the CAP PT Program and pediatric reference ranges employing different immunoassays indicated the probability of lack of specificity for T4 and T3 immunoassay tests. To adequately assess this phenomenon, the isotope dilution tandem mass spectrometric method was developed as described in this example. Serum T4 and T3 detection methods have evolved through a variety of technologies since the 1950s. Radioimmunoassay (RIA) methods to detect thyroid hormones were developed in the 1970s. Serum T4 and T3 concentrations are currently measured by competitive immunoassay methods (IAs) that are mostly non-isotopic and use enzymes, fluorescence or chemiluminescence molecules as signals [27]. Table 2 clearly indicates that current IAs for T4 and T3 lack specificity and give mean results differing by a factor of approximately 2 in the College of American Pathologists Proficiency Testing (CAP PT) programs. Total hormone assays necessitate the inclusion of a displacing agent (such as salicylate) to release the hormone from its binding proteins [28]. The displacement of hormone binding from serum proteins by such agents, together with the large sample dilution employed in modern assays, facilitates the binding of hormone to the antibody reagent.
  • Since T3 is ten-fold lower in concentration compared with T4 in blood it therefore presents both a technical sensitivity and precision challenge despite the use of a higher specimen volume. Although a reliable high-range T3 measurement is critical for diagnosing hyperthyroidism, a reliable normal-range measurement is also important for adjusting antithyroid drug dosage and detecting hyperthyroidism in sick hospitalized patients, in whom a paradoxically normal T3 value may indicate hyperthyroidism.
  • The correlation coefficient for the T4 comparisons (0.931) is significantly better than for the T3 comparisons (0.848) (Figures 4 and 5). T3 by tandem mass spectrometry gave slightly higher results than those obtained by the DPC Immulite™ (Figure 4). While this is true for children, preliminary data for non-pregnant and pregnant women indicates a very poor correlation for T3 in both groups (r between 0.407-0.574) (i.e. there is a poor correlation between DPC Immulite and the method of the present teaching in both non-pregnant and pregnant women).
  • The reasons for this are not clear but could include standardization issues, heterophilic antibodies, etc. Of importance, reverse T3, which lacks a daughter ion of 127 m/z, does not interfere in the tandem mass spectrometry methods. Applying the tandem mass spectrometric method to CAP PT samples in the K/KN (thyroid) general ligand program again revealed that around 85% of the immunoassay methods for T3 gave means on samples which were lower than the means obtained by the tandem mass spectrometry methods of this applicant's teaching while 15% had higher means. For T4, the tandem mass spectrometry method resulted in lower means than those of the immunoassay methods.
  • In conclusion, correlations between immunoassays and tandem mass spectrometry for T4 and T3 have been demonstrated. The correlation is better for T4 than for T3. Further, the correlation is less impressive during pregnancy. Recovery studies from several different sera using deuterated T4 as internal standard showed consistent (90-109%) recoveries for both T4 and T3 (Tables 7 and 8). The recovery differences found between samples were surprisingly larger for T4 than for T3. This indicates a lack of need to use deuterated T3 as the T3 internal standard. The isotope dilution tandem mass spectrometric method of the applicant's teaching is rapid (less than 7 minutes), accurate (provides the true result as has been assessed by recovery studies), specific (measures only the analyte it purports to measure), precise (low %CV) and easy to perform. Table 3: Tandem mass-spectrometer working parameters
    Parameter Value
    (Nebulizer gas (NEB) 8
    Curtain gas (CUR) 10
    Collision gas (CAD) 6
    TurboIon Spray Heater gas 7 L/min
    TurboIon Spray (IS) voltage 4500 V
    Entrance Potential (EP) 7.5 V
    Collision cell Exit Potential (CXP) 5 V
    Source temperature 450°
    Dwell time 250 msec
    Table 4: Gradient parameters
    Time (min) Methanol (%)
    3.50 75
    5.25 76
    5.50 100
    7.00 End
    Table 5: Within day precision (n=10)
    Analyte CONTROL 1 CONTROL 2
    Mean (ng/mL) SD CV (%) Mean (ng/mL) SD CV (%)
    T3 1.04 0.014 1.36 2.44 0.077 3.19
    T4 24.1 0.437 1.81 81.2 1.502 1.85
    Table 6: Between day precision (n=20,1 run per day for 20 days)
    Analyte CONTROL 1 CONTROL 2 CONTROL 3
    Mean (ng/mL) SD CV (%) Mean (ng/mL) SD CV (%) Mean (ng/mL) SD CV (%)
    T3 1.08 0.05 4.47 239 0.22 9.21 3.49 0.31 9.00
    T4 24.4 1.39 5.69 76.6 3.11 4.06 116.3 4.15 3.57
    Table 7: Recovery of added thyroxine (T4)
    Sample # Added (ng/mL) Detected mean Added amount recovered Recovery, %
    1 (n=8) 0 85.9 NA* NA
    10 96.7 10.8 108.0
    40 127.5 41.6 104.0
    2 (n=5) 0 62.6 NA NA
    10 72.1 9.5 95.0
    40 98.0 35.4 90.0
    3 (n=5) 0 73.8 NA NA
    10 84.7 10.9 109.0
    40 116 42.2 105.0
    4 (n=5) 0 58.3 NA NA
    10 68.0 9.7 97.0
    40 95.0 36.7 92.0
    *NA - not applicable
    Table 8: Recovery of added triiodothyronine (T3)
    Sample # Added (ng/mL) Detected mean Added amount recovered Recovery, %
    1 (n=8) 0 1.88 NA NA
    0.25 2.12 0.24 96.0
    1.00 2.85 0.97 97.0
    2 (n=5) 0 1.70 NA NA
    0.25 1.96 0.26 104.0
    1.00 2.76 1.06 106.0
    3 (n=5) 0 1.56 NA NA
    0.25 1.81 0.25 100.0
    1.00 2.62 1.06 106.0
    4 (n=5) 0 0.49 NA NA
    0.25 0.74 0.25 100.0
    1.00 1.50 1.01 101.0
    *NA - not applicable
    • 4. Analysis of free thyroxine (FT4)
  • Most routine clinical chemistry service laboratories provide for the measurement of free thyroxine (FT4) by an analogue (direct) method with 24 hours and 7 day per week availability. Nevertheless, the validity of analogue FT4 immunoassays has long been questioned and patient's results using this approach frequently do not fit in with the clinical picture. Because of this, direct free T4's that are below the 2.5th percentile and many that are above the 97.5th percentile are often sent for further measurement by the current "gold standard" method for FT4, equilibrium dialysis. In approximately 50% of these cases the FT4 by equilibrium dialysis has been found to be normal. The present methods teach a rapid, reliable free T4 method employing isotope dilution tandem mass spectrometry and compares results obtained by this method with both the analogue (direct) free T4 and the time-consuming and relatively expensive equilibrium dialysis procedures.
    • Methods: Chemicals and reagents
  • Thyroxine (T4) was purchased from Sigma (St Louis, MO). A stable deuterium-labeled internal standard, L-thyroxin-d2 was synthesized according to procedures described in the literature (29, 30) by Dr Tomas Class from the Chemistry Department at Georgetown University. HPLC grade methanol was purchased from VWR Scientific. All other chemicals were of analytical grade and were purchased from Sigma.
    • Solutions and standards
  • Stock solutions of T4 and internal standard (IS) were prepared separately to obtain concentration of 10 mg/mL for each using 40% ammonium hydroxide (v/v) in methanol as a solvent. The analyte stock solutions were diluted with methanol to obtain the spiking solutions. The solutions were stored at -20°C and could be used for several months. Standards for the T4 calibration curve in the range of 2.5-50 pg/mL were prepared by adding the analytes to water. A solution of 0.05 ng/mL d2-T4 in methanol was used as internal standard.
    • Sample preparation
  • Serum or plasma samples were obtained from greater than 42 healthy pregnant and 29 non-pregnant women in a study approved by the Institutional Review Board (IRB) and were thawed at room temperature. 0.6 ml samples were filtered through Centrifree YM-30 ultrafiltration devices (30,000 MW cut-off, Millipore, Bedford, MA) by centrifugation employing the Eppendorf temperature controlled centrifuge (model # 5702 R, Eppendorf, AG, Hamburg) and using a fixed angle rotor at 2900 rpm and a temperature of 25° for 1 hour. 180 µL IS [0.05ng/mL] was added to 360 µL ultrafiltrate and 400 µL was injected onto the C-18 column of the LC/MS/MS system. This ultrafiltration process replaces the dialysis step of the classic equilibrium dialysis method. The ultrafiltration step includes removal of all proteins having a molecular weight of greater than 30,000. The liquid chromatography step can be used to further separate and purify the hormone.
    • LC/MS/MS Setup
  • An API 4000™ tandem mass-spectrometer (SCIEX, Toronto, Canada) equipped with TurboIonSpray and Agilent 1100 HPLC system was used to perform the analysis. Negative ion multiple reaction-monitoring (MRM) mode was used. The transitions to monitor were selected and are m/z 775.9→126.9 for T4, m/z 777.9→126.9 for d2-T4. Nitrogen served as auxiliary, curtain and collision gas. Gas flow rates, source t°, Ion Spray voltages and collision energies were optimized for every compound by infusion of 1 µg/mL standards solutions in methanol at 20 µL/min and by flow-injection analysis (FIA) at LC flow rate. The main working parameters of mass spectrometer used are summarized in Table 9. Data processing was performed on Analyst 1.4.1 software package. Although the negative mode was used in this example, a positive mode can be used but is less sensitive.
    • LC-MS-MS procedure
  • The procedure used is based on an online extraction/cleaning of the injected samples with subsequent introduction into the mass-spectrometer by using a built-in Valco switching valve. 400 µL of the sample was injected onto the Supelco LC-18-DB (3.3 mm x 3.0 mm, 3.0 µm ID) chromatographic column equipped with a Supelco Discovery C-18 (3.0 mm) guard column, where it underwent cleaning with 20% (v/v) methanol in 5 mM ammonium acetate pH-4.0 at flow rate 0.8 mL/min. After 4 minutes of cleaning the switching valve was activated, the column was flushed with a water/methanol gradient at flow rate of 0.6 mL/min and the samples were introduced into the mass-spectrometer. The gradient parameters that were used are shown in Table 10. The free T4 chromatogram is shown in Figure 6.
    • Equilibrium dialysis
  • The Nichols free T4 kit (Nichols Institute Diagnostics, Catalogue # 30-0652, San Clemente, CA) was used according to the directions provided by the manufacturer. A comparison between the equilibrium dialysis and the tandem mass spectrometric method were performed on patient samples (n=68).
    • Analogue/direct free T4
  • The Dade RxL Dimension was used for the direct free T4 method.(Dade-Behring Diagnostics, Glasgow, DE).Results on patient samples were compared with values obtained using tandem mass spectrometry (n=154).
    • Between-day and within-day precision
  • The between-day and within-day precision was assessed at 3 different concentrations (Table 12).
    • Results and Discussion
  • Tables 9 and 10 provide the analytical parameters employed for the tandem mass spectrometric method. Figure 6 shows a typical chromatogram for free T4 measured by tandem mass spectrometry using the method described. The time per analysis is approximately 8.5 minutes although a steeper gradient could shorten this to about 6 minutes. The Eppendorf centrifuge allows for the centrifugation of 30 tubes simultaneously so that the total run time for 30 patient samples at the 25°C temperature used is 1 hour plus 3 hours and 15 minutes, or 4 hours and 15 minutes. This ultrafiltration plus LGMS/MS assay is considerably quicker than the time consuming equilibrium dialysis method. The latter requires 16-18 hour dialysis at 37°C followed by an immunoassay and therefore the turn-around-time is several days. Also, very few laboratories in North America provide the equilibrium dialysis approach. The concentration of FT4 is temperature dependent (31). If the centrifugation of the Amicon Centrifree tubes occurs at 25°C (see Figure 7 and Table 11) the results obtained by the tandem mass spectrometric method closely correlate with those obtained by equilibrium dialysis, which employs a temperature of 37°C. This 12°C temperature difference is probably the result of different membranes being employed in the equilibrium dialysis and ultrafiltration methods. The correlation between the new isotope dilution tandem mass spectrometric method and the conventional gold standard equilibrium dialysis method was excellent. Equilibrium dialysis= 0.971 Mass Spectrometry+0.041, n=68, Syx=1.381, r=0.954 (Figure 8). In contrast a poor correlation was found with the analogue (direct) FT4 method (Immunoassay=0.326 Mass Spectrometry+6.27, n=154, Syx=1.96, r=0.459, Figure 9). The between-day and within-day precision shows all concentrations tested gave coefficient of variations (Cvs) of less than 7.1 % (Table 12). This performance is superior to that obtained using the difficult equilibrium dialysis method. The lower limit of detection (a reading greater than three standard deviations over the baseline noise) is 2.5 pg/mL.
  • These studies confirm that the analogue procedures give poor results for free T4 which is further supported when reflex testing for all FT4s below the 2.5th percentile and all FT4s above the 97.5th percentile which also have normal thyroid-stimulating hormone (TSH) values is done. Approximately, 50% of these free T4s run on either the Dade RxL Dimension™or the DPC Immulite™ give normal results when run by equilibrium dialysis. Finally in the present study, 80% of FT4s greater than the 96.7th percentile by tandem MS are associated with TSHs of less than 1.0 uIU/mL (the latter measured by the Dade RxL Dimension™) while in the same cohort of patients, only 40% of FT4s greater than the 96.7th percentile measured by direct IA had TSHs of less than 1.0 uIU/mL.
  • It should also be noted that prior to using tandem mass spectrometry on the plasma ultrafiltrate, attempts were made to measure FT4 on the ultrafiltrate by IA using several approaches which included an RIA kit (Nichols), the Dade RxL™ and DPC IMMULITE™ platforms. In all cases results were exceedingly low indicating that this was not a viable alternative.
  • In conclusion, a new isotope dilution tandem mass spectrometric method for the measurement of FT4 employing ultrafiltration has been developed. The procedure has excellent precision, compares well with the gold standard. Based on these attractive characteristics this method of FT4 measurement will have a wide applicability in the clinical setting. Table 9. Tandem mass-spectrometer working parameters
    Parameter Value
    Curtain gas (CUR) 14
    Gas 1 (Nebulizer gas) 45
    Gas2 (Heater gas) 20
    CAD gas 12
    Turbolon Spray (IS) voltage - 4500 V
    Entrance Potential (EP) -10V
    Collision cell Exit Potential (CXP) -4V
    Source t 650°
    Dwell time 250 msec
    Table 10. Gradient parameters
    Time Methanol (%)
    0.0 10
    2.5 20
    3.5 20
    3.6 95
    4.5 99
    5.9 100
    Table 11: Effect of temperature on free T4 and on FT4/TT4 ratios
    Free T4 (pg/mL)
    LC/MS1MS after ultrafiltration Equilidrium dialysis after Eq.dial
    Plasma 4 C 20 C 25 C 40 C
    40598 4.88 7.54 11.80 19.63 11.57 12.45
    06409 4.85 7.36 12.67 19.43 10.77 12.05
    09287 3.62 6.17 8.26 14.23 6.94 8.32
    53230 8.06 15.53 19.97 34.57 21.11 20.80
    46537 5.26 13.03 13.80 25.90 14.17 13.70
    40620 7.87 11.50 9.69 17.00 9.42 10.40
    *MS Dial - samples running on MS after dialysis
    Free T4/ Total T4*
    LC/MS/MS after ultrafiltration Equilidrium dialysis LC/MS/MS after Eq.dial
    Plasma 4C 20 C 25 C 40 C
    40598 0.067 0.104 0.163 0.271 0.160 0.172
    06409 0.086 0.131 0.225 0.345 0.191 0.214
    09287 0.074 0.126 0.169 0.291 0.142 0.170
    53230 0.166 0.319 0.410 0.710 0.433 0.427
    46537 0.140 0.347 0.368 0.690 0.378 0.365
    40620 0.158 0.232 0.195 0.342 0.190 0.209
    * Free T4 (pg/mL), Total T4 (ng/mL)
    Table 12: Within-day and Between-day precision
    Within-day (n=10) Between-day (n=20)
    Control Mean (pg/mL) CV (%) Mean (pg/mL) CV (%)
    Low 6.6 4.1 6.6 7.1
    Medium 12.7 6.4 12.8 7.1
    High 26.2 6.6 24.4 6.7
  • This demonstrates a simple method for preparing and detecting FT4 by mass spectrometry.
    • 5. Analysis of thyroid hormones and steroid hormones
  • A sample of 500 to 1000 µL of plasma is used. Proteins are precipitated with 150 µL of acetonitrile and vortexed. The sample is centrifuged, and 200 µL of the supernatant is injected onto a C-18 column coupled to a tandem mass spectrometer (LC/MS/MS). The column is washed with 20% methanol in 5mM ammonium acetate for 3 minutes. The valve on the column is switched and the sample is eluted in a methanol gradient of 20 to 100%. The total run time is 10 minutes. Slight adjustments to the volumes, concentrations and times described can be made, as is known to those skilled in the art.
  • A sample of the eluant is introduced into an ion-spray ionization chamber and analyzed by API 3000™ mass spectrometer using the negative mode for thyroid hormones in the sample. Steroid hormones in the sample are ionized by photoionization, with the spectrometer in the negative or positive mode. Analysis in the positive mode is typically made for DHEA, Aldosterone, Cortisol, 11-Deoxycortisol, Androstenedione, Testosterone, Estradiol, 17-OH Progesterone, Progesterone, Allopregnalone, Vitamin D, 25,hydroxyl Vitamin D, 1,25 dihydroxy Vitamin D, corticosterone and aldosterone, whereas analysis in the negative mode is typically made for 16-OH Estrone, 2-OH Estrone, Estriol and DHEAS. However, it is possible to analyze any of the hormones in either positive or negative mode.
  • This demonstrates a simple method of preparing a complex biological matrix for analysis of possible steroid and thyroid hormone content. Steroid hormones which are run in the negative mode can be run simultaneously with the thyroid hormones.
  • The results indicate that this technique, allows for the identification and characterization of low levels of thyroid hormone in human plasma and saliva.
    • 6. Analysis of FT3 hormone
  • FT3 was analyzed by the same method as FT4 (Example 4), except for the analysis of the same transition ions for total T3 and using the API 5000™ mass spectrometer.
    • 7. Simultaneous analysis of FT4 and FT3
  • Patients with either hyperthyroidism or hypothyroidism require frequent assessment of thyroid function through measurement of their FT4 and FT3 concentrations. Further, people with thyroid ablation require thyroid replacement therapy, such as synthroid. Measurement of their FT4 and FT3 concentrations is important when assessing their dosage regimen. Accordingly, an efficient assay method for the simultaneous analysis of FT3 and FT4 is beneficial.
  • FT4 and FT3 were analyzed simultaneously by a similar method of Example 4 except using the API 5000™ mass spectrometer. 100µL mixture of T3 (25pg/mL) and T4 (1 ng/mL) with internal standard T4-d2 were injected onto the column by autosampler, and the column was washed by 20% MeOH buffer for 2 minutes. Gradient elution started from 20% MeOH to 100% MeOH in 2 minutes after the Valco valve was activated at 2 minutes, and then kept at 100% for another 2 minutes. The retention times were: T3, 4.34 minutes, T4, 4.60 minutes, and T4-d2, 4.61 minutes. Figure 10 shows the mass spectrums of the analytes. Standard curves for FT3 (1-25 pg/ml) and FT4 (5-50 pg/ml) can be run with the analysis of the samples.
  • While the applicant's teachings are described in conjunction with various embodiments, it is not intended that the applicant's teachings be limited to such embodiments. On the contrary, the applicant's teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.
    • REFERENCES
  • All references listed herein are incorporated by reference in their entirety.
    1. 1. Lum SM, Nicoloff JT, Spencer CA, Kaptein EM. Peripheral tissue mechanism for maintenance of serum triiodothyronine values in a thyroxine-deficient state in man. J Clin Invest 1984; 73(2):570-575.
    2. 2. Sakata S, Nakamura S, Miura K. Autoantibodies against thyroid hormones or iodothyronine. Implications in diagnosis, thyroid function, treatment, and pathogenesis. Ann Intern Med 1985; 103(4):579-589.
    3. 3. Beck-Peccoz P, Romelli PB, Cattaneo MG, Faglia G, White EL, Barlow JW, Stockigt JR. Evaluation of free thyroxine methods in the presence of iodothyronine-binding autoantibodies. J Clin Endocrinol Metab 1984; 58(4):736-739.
    4. 4. Klee GG. Human anti-mouse antibodies. Arch .
    5. 5. College of American Pathologists Proficiency Survey Report on Y-03, RAP-03 and K-06 specimens for 2003.
    6. 6. Soldin SJ. Digoxin--issues and controversies. Clin Chem 1986; 32(1 Pt 1):5-12.
    7. 7. Soldin SJ, Papanastasiou-Diamandi A, Heyes J, Lingwood C, Olley P. Are immunoassays for digoxin reliable? Clin Biochem 1984; 17(5):317-320.
    8. 8. Thong B, Soldin SJ, Lingwood CA. Lack of specificity of current anti-digoxin antibodies, and preparation of a new, specific polyclonal antibody that recognizes the carbohydrate moiety of digoxin. Clin Chem 1985; 31(10):1625-1631.
    9. 9. Murthy JN, Davis DL, Yatscoff RW, Soldin SJ. Tacrolimus metabolite cross-reactivity in different tacrolimus assays. Clin Biochem 1998; 31(8):613-617.
    10. 10. Murthy JN, Yatscoff RW, Soldin SJ. Cyclosporine metabolite cross-reactivity in different cyclosporine assays. Clin Biochem 1998; 31(3):159-163.
    11. 11. Shen S, Elin RJ, Soldin SJ. Characterization of cross reactivity by .
    12. 12. Ghoshal AK, Soldin SJ. tacrolimus II assay: is it reliable at low blood concentrations? A comparison with tandem MS/MS. Clin Biochem 2002; 35(5): 389-392.
    13. 13. Soldin SJ, Steele BW, Witte DL, Wang E, Elin RJ. Lack of specificity of cyclosporine immunoassays. Results of a College of American Pathologists Study. Arch Pathol Lab Med 2003; 127(1):19-22.
    14. 14. Despres N, Grant AM. Antibody interference in thyroid assays: a potential for clinical misinformation. Clin Chem 1998; 44(3):440-454.
    15. 15. Same DH, Refetoff S, Nelson JC, Linarelli LG. A new inherited abnormality of thyroxine-binding globulin (TBG-San Diego) with decreased affinity for thyroxine and triiodothyronine. J Clin Endocrinol Metab 1989; 68(1): 114-119.
    16. 16. Burman KD, Bongiovanni R, Garis RK, Wartofsky L. Boehm TM. Measurement of serum T4 concentration by high performance liquid chromatography. J Clin Endocrinol Metab, 1981; 53(5): 909-912.
    17. 17. Tai SS, Sniegoski LT, Welch MJ. Candidate reference method for total thyroxine in human serum: use of isotope-dilution liquid chromatography-mass spectrometry with electrospray ionization. Clin Chem 2002; 48(4):637-642.
    18. 18. Thienpont LM, De Brabandere VI, Stockl D, De Leenheer AP. Development of a new method for the determination of thyroxine in serum based on isotope dilution gas chromatography mass spectrometry. Biol Mass Spectrom 1994; 23(8): 475-482.
    19. 19. Thienpont LM, Fierens C, De Leenheer AP, Przywara L. Isotope dilution-gas chromatography/mass spectrometry and liquid chromatography/electrospray ionization-tandem mass spectrometry for the determination of triiodo-L-thyronine in serum. Rapid Commun Mass Spectrom 1999; 13(19):1924-1931.
    20. 20. De Brabandere VI, Hou P, Stockl D, Thienpont LM, De Leenheer AP. Isotope dilution-liquid chromatography/ electrospray ionization-tandem mass spectrometry for the determination of serum thyroxine as a potential reference method. Rapid Commun Mass Spectrom 1998; 12(16): 1099-1103.
    21. 21. Ramsden, D.B. and M.J. Farmer, Development of a gas chromatographic selected ion monitoring assay for thyroxine (T4) in human serum. Biomed Mass Spectrom 1984; 11(8):421-427.
    22. 22. Nishinaga A, Cahnmann HJ, Kon H, Matsuura T. Model reactions for the biosynthesis of thyroxine. XII. The nature of a thyroxine precursor formed in the synthesis of thyroxine from diiodotyrosine and its keto acid analog. Biochemistry 1968; 7(1):388-397.
    23. 23. Choi MH, Kim, JN, Chung BC. Rapid HPLC-Electrospray Tandem Mass Spectrometric Assay for Urinary Testosterone and Dihydrosterone Glucuronides from Patients with Benign Prostate Hyperplasia. Clin Chem 2003;49(2): 22-325.
    24. 24. Biancotto G, Angeletti R, Traldi P, Silvestri MS, Guidugli F. Determination of 17β-Estradiol in Bovine Plasma: Development of a Highly Sensitive Technique by Ion Trap Gas Chromatography-Tandem Mass Spectrometry Using Negative Ion Chemical Ionization. J Mass Spectrom 2002;37: 1226-1271.
    25. 25. Lai CC, Tsai CH, Tsai FJ, Wu JY, Lin WD, Lee CC. Rapid Screening Assay of Congenital Adrenal Hyperplasia by Measuring 17α-Hydroxy- progesterone with High-Performance Liquid Chromatography/ Electrospray Ionization Tandem Mass Spectrometry From Dried Blood Spots. J Clin Lab Anal 2002;16: 20-25.
    26. 26. Vierhapper H, Nowotny P, Waldausl W. Reduced Production Rates of Testosterone and Dihydrosterone in Healthy Men Treated with Rosiglitazone. .
    27. 27. Nelson JC, Wilcox RB. Analytical performance of free and total thyroxine assays. Clin Chem 1996; 42(1):146-154.
    28. 28. Evans SE, Burr WA, Hogan TC. A reassessment of 8-anilino-1-naphthalene sulphonic acid as a thyroxine binding inhibitor in the radioimmunoassay of thyroxine. Ann Clin Biochem 1977; 14(6):330-334.
    29. 29. Ekins R. Validity of Analogue Free Thyroxin Immunoassays. Clin Chem 1987; 33:2137-52.
    30. 30. Tai SS, Sniegoski LT, Welch MJ. Candidate reference method for total thyroxine in serum: use of isotope-dilution liquid chromatography-mass spectrometry with electrospray ionization. Clin Chem 2002; 48: 637-42.
    31. 31. van der Sluijs Veer G., Vermes I, Bonte HA, Hoorn RKJ. Temperature effects on Free-Thyroxine Measurements: Analytical and Clinical Consequences. Clin Chem 1992; 38:1327-31.

Claims (25)

  1. A method for mass spectrometric analysis of a sample containing or suspected of containing free thyroxine (FT4) hormone, comprising the steps:
    (a) providing a sample containing or suspected of containing FT4 hormone;
    (b) separating FT4 hormone from the sample and from bound T4 by centrifugation using an ultrafiltration device;
    (c) collecting FT4 hormone separated from bound T4; and
    (d) analyzing FT4 hormone separated from bound T4 using a mass spectrometer.
  2. The method of claim 1 further comprising analysis of free triiodothyronine (FT3) hormone, wherein the method comprises the steps of:
    (a) providing the sample containing or suspected of containing FT4 and FT3;
    (b) separating FT4 hormone and FT3 hormone from the sample and from bound T4 and bound T3;
    (c) collecting FT4 and FT3 hormone separated from bound T4 and bound T3; and
    (d) analyzing FT4 and FT3 hormone separated from bound T4 and bound T3 using a mass spectrometer.
  3. The method according to claim 2 wherein the hormones are analyzed simultaneously.
  4. The method according to claim 2 wherein the hormones are analyzed sequentially.
  5. The method according to claim 2 wherein the hormones are analyzed by isotope dilution tandem mass spectrometry.
  6. The method according to claim 1 or 2 wherein the sample is obtained from a biological sample selected from blood, plasma, serum, urine and saliva, or any combination thereof.
  7. The method of claim 6 wherein the biological sample is blood.
  8. The method of claim 6 wherein the biological sample is plasma.
  9. The method of claim 6 wherein the biological sample is serum.
  10. The method of claim 6 wherein the biological sample is urine.
  11. The method of claim 6 wherein the biological sample is saliva.
  12. The method according to claim 1 or 2 wherein size of said sample containing or suspected of containing said hormone(s) is at least about 500 µL.
  13. The method of claim 1 or 2 wherein the step of separating the hormone(s) from the sample and from bound T4 and/or bound T3 comprises use of ultrafiltration device using a mesh size of approximately 30,000 MW
  14. The method of claim 13 wherein the step of separating the hormone(s) from the sample and from bound T4 and/or bound T3 comprises use of Centrifree YM-30™ ultrafiltration device.
  15. The method of any of claims 13 or 14 wherein the hormone(s) is/are separated by centrifugation at about 25°C for about 1 hour.
  16. The method according to claim 1 or 2 wherein the step of separating the hormone(s) from the sample and from bound T4 and/or bound T3 further comprises an on-line extraction and a built-in switch valve.
  17. The method according to claim 1 or 2 wherein the mass spectrometer is a liquid chromatography-tandem-mass spectrometer.
  18. The method according to claim 17 wherein the liquid chromatographytandem mass spectrometer is equipped with an electrospray ionization source.
  19. The method according to claim 1 or 2 wherein said step of analyzing the hormone(s) using a mass spectrometer comprises an ionization technique selected from photoioinization, electrospray ionization, atmospheric pressure chemical ionization, and electron capture ionization.
  20. The method according to claim 19 wherein said ionization technique is electrospray ionization.
  21. The method according to claim 20 wherein said ionization is performed in negative mode.
  22. The method according to claim 1 or 2 wherein said step of analyzing the hormone(s) using a mass spectrometer comprises multiple reaction monitoring.
  23. The method according to claim 1 or 2 wherein said step of analyzing the hormone(s) using a mass spectrometer comprises selected ion monitoring.
  24. The method of any one of claims 1 to 23 wherein the mass spectrometer is API 4000™.
  25. The method of any one of claims 1 to 23 wherein the mass spectrometer is API 5000™.
EP05851243A 2005-03-31 2005-10-20 Free thyroxine and free triiodothyronine analysis by mass spectrometry Active EP1872120B1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US66671205P 2005-03-31 2005-03-31
PCT/US2005/038232 WO2006107339A2 (en) 2005-03-31 2005-10-20 Free thyroxine and free triiodothyronine analysis by mass spectrometry

Publications (3)

Publication Number Publication Date
EP1872120A2 EP1872120A2 (en) 2008-01-02
EP1872120A4 EP1872120A4 (en) 2009-04-29
EP1872120B1 true EP1872120B1 (en) 2012-12-05

Family

ID=37073885

Family Applications (1)

Application Number Title Priority Date Filing Date
EP05851243A Active EP1872120B1 (en) 2005-03-31 2005-10-20 Free thyroxine and free triiodothyronine analysis by mass spectrometry

Country Status (5)

Country Link
US (1) US8227259B2 (en)
EP (1) EP1872120B1 (en)
JP (1) JP2008534955A (en)
CA (1) CA2608796C (en)
WO (1) WO2006107339A2 (en)

Families Citing this family (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7745226B2 (en) 2005-04-06 2010-06-29 Quest Diagnostics Investments Incorporated Methods for detecting vitamin D metabolites
WO2007090081A2 (en) * 2006-01-27 2007-08-09 The Board Of Governors For Higher Education State Of Rhode Island And Providence Plantations Analysis of mycophenolic acid in saliva using liquid chromatography tandem mass spectrometry
CA2654319C (en) * 2006-05-26 2014-08-12 Laboratory Corporation Of America Holdings Liquid chromatography with tandem mass spectrometry of estrone, estradiol and free thyroxine
US20080102535A1 (en) * 2006-11-01 2008-05-01 Chace Donald H Measuring thyroxine levels from dried blood samples using mass spectrometry
US20090134325A1 (en) * 2007-11-27 2009-05-28 Goldman Mildred M Methods for detecting estradiol by mass spectrometry
US7972868B2 (en) 2007-11-28 2011-07-05 Quest Diagnostics Investments Incorporated Methods for detecting dihydroxyvitamin D metabolites by mass spectrometry
US8030084B2 (en) 2007-12-06 2011-10-04 Quest Diagnostics Investments Incorporated Thyroglobulin quantitation by mass spectrometry
US8916385B2 (en) 2007-12-13 2014-12-23 Quest Diagnostics Investments, Inc. Methods for detecting estrone by mass spectrometry
US7977117B2 (en) 2009-12-03 2011-07-12 Quest Diagnostics Investments Incorprated Vitamin D metabolite determination utilizing mass spectrometry following derivatization
WO2011072168A1 (en) 2009-12-11 2011-06-16 Quest Diagnostics Investments Incorporated Mass spectrometric determination of cookson-derivatized, non-metabolized vitamin d
CN107607662B (en) 2009-12-11 2020-05-08 奎斯特诊断投资公司 Mass spectrometry of steroids in multiplex samples
US8669519B2 (en) 2011-12-05 2014-03-11 Quest Diagnostics Investments, Inc. Methods for detecting reverse triiodothyronine by mass spectrometry
WO2014047316A1 (en) 2012-09-20 2014-03-27 Quest Diagnostics Investments Incorporated Thyroglobulin quantitation by mass spectroscopy
WO2016019037A1 (en) * 2014-07-29 2016-02-04 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Free hormone and hormone metabolite workup and analysis by mass spectrometry
JP6337970B2 (en) * 2014-09-17 2018-06-06 株式会社島津製作所 Mass spectrometer
JP2020526811A (en) 2017-07-07 2020-08-31 エヌチェーン ホールディングス リミテッドＮｃｈａｉｎ Ｈｏｌｄｉｎｇｓ Ｌｉｍｉｔｅｄ Control flow in blockchain script
CN111398490A (en) * 2020-03-04 2020-07-10 上海睿质科技有限公司 Kit for detecting free triiodothyronine and free thyroxine by mass spectrometry
CN113281526B (en) * 2021-03-18 2022-04-08 杭州微策生物技术股份有限公司 Sample pad treatment reagent of free thyroxine detection reagent strip
CN113390977B (en) * 2021-04-13 2023-05-26 杭州凯莱谱精准医疗检测技术有限公司 Method for simultaneously measuring free thyroid hormones T3, rT3, T4 and cortisol in saliva
CN116626191A (en) * 2022-03-22 2023-08-22 合肥歆智医疗器械有限公司 Ultrafiltration-liquid chromatography tandem mass spectrometry for detecting free testosterone

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6153440A (en) * 1998-09-23 2000-11-28 The Regents Of The University Of California Simultaneous measurement of free triiodothyronine and free thyroxine by equilibrium dialysis and immunoassay

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4023398A (en) 1975-03-03 1977-05-17 John Barry French Apparatus for analyzing trace components
US4328420A (en) 1980-07-28 1982-05-04 French John B Tandem mass spectrometer with open structure AC-only rod sections, and method of operating a mass spectrometer system
US4741897A (en) 1986-07-08 1988-05-03 Baxter Travenol Thyroxine analogs and reagents for thyroid hormone assays
CA1307859C (en) 1988-12-12 1992-09-22 Donald James Douglas Mass spectrometer and method with improved ion transmission
US5179278A (en) 1991-08-23 1993-01-12 Mds Health Group Limited Multipole inlet system for ion traps
US5248875A (en) 1992-04-24 1993-09-28 Mds Health Group Limited Method for increased resolution in tandem mass spectrometry
US5412208A (en) 1994-01-13 1995-05-02 Mds Health Group Limited Ion spray with intersecting flow
WO1997007530A1 (en) 1995-08-11 1997-02-27 Mds Health Group Limited Spectrometer with axial field
AU2001264650A1 (en) 2000-05-18 2001-11-26 Mayo Foundation For Medical Education And Research Adrenal dysfunction
AUPR195900A0 (en) * 2000-12-08 2001-01-04 Women's And Children's Hospital Detection of compounds such as thyroxine
CA2519626C (en) * 2003-04-14 2013-06-18 Georgetown University Thyroid hormone analysis by mass spectrometry with minimal sample preparation

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6153440A (en) * 1998-09-23 2000-11-28 The Regents Of The University Of California Simultaneous measurement of free triiodothyronine and free thyroxine by equilibrium dialysis and immunoassay

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CLARKE WILLIAM ET AL: "Analysis of free hormone fractions by an ultrafast immunoextraction/displacement immunoassay: studies using free thyroxine as a model system.", 11 February 2005, ANALYTICAL CHEMISTRY 15 MAR 2005 LNKD- PUBMED:15762597, VOL. 77, NR. 6, PAGE(S) 1859 - 1866, ISSN: 0003-2700 *

Also Published As

Publication number Publication date
WO2006107339A3 (en) 2007-11-01
CA2608796A1 (en) 2006-10-12
EP1872120A4 (en) 2009-04-29
CA2608796C (en) 2013-09-03
JP2008534955A (en) 2008-08-28
WO2006107339A2 (en) 2006-10-12
US8227259B2 (en) 2012-07-24
US20060223188A1 (en) 2006-10-05
EP1872120A2 (en) 2008-01-02

Similar Documents

Publication Publication Date Title
USRE44401E1 (en) Thyroid hormone analysis by mass spectrometry
EP1872120B1 (en) Free thyroxine and free triiodothyronine analysis by mass spectrometry
US7473560B2 (en) Steroid hormone analysis by mass spectrometry
Soldin et al. The measurement of free thyroxine by isotope dilution tandem mass spectrometry
US20220326260A1 (en) Methdos for detecting estradiol by mass spectrometry
US7935921B2 (en) Methods and systems for the quantitative analysis of biomarkers
Carvalho The coming of age of liquid chromatography coupled to tandem mass spectrometry in the endocrinology laboratory
Kulle et al. Principles and clinical applications of liquid chromatography—tandem mass spectrometry for the determination of adrenal and gonadal steroid hormones
Ackermans et al. LC–MS/MS in endocrinology: what is the profit of the last 5 years?
US9012835B2 (en) Methods for simultaneous quantification of thyroid hormones and metabolites thereof by mass spectrometry
US11536733B2 (en) Methods and systems for the detection of 11-oxo androgens by LC-MS/MS
WO2016019037A1 (en) Free hormone and hormone metabolite workup and analysis by mass spectrometry
Field Tandem mass spectrometry in hormone measurement
Zhang et al. Detection and quantification of 3, 5, 3′-triiodothyronine and 3, 3′, 5′-triiodothyronine by electrospray ionization tandem mass spectrometry
Wu et al. Simultaneous measurement of 19 steroid hormones in dried blood spots using ultra-performance liquid chromatography-tandem mass spectrometry
Stanczyk Androgen measurements: methods, interpretation, and limitations
Stanczyk Androgen measurements
Holterhus Principles and clinical applications of liquid chromatography-tandem mass spectrometry for the determination of adrenal and gonadal steroid hormones

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20071017

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL BA HR MK YU

DAX Request for extension of the european patent (deleted)
A4 Supplementary search report drawn up and despatched

Effective date: 20090401

RIC1 Information provided on ipc code assigned before grant

Ipc: G01N 33/78 20060101AFI20090323BHEP

17Q First examination report despatched

Effective date: 20090803

REG Reference to a national code

Ref country code: DE

Ref legal event code: R079

Ref document number: 602005037345

Country of ref document: DE

Free format text: PREVIOUS MAIN CLASS: G01N0033000000

Ipc: G01N0033680000

RIC1 Information provided on ipc code assigned before grant

Ipc: G01N 33/78 20060101ALI20120308BHEP

Ipc: G01N 33/68 20060101AFI20120308BHEP

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI SK TR

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: AT

Ref legal event code: REF

Ref document number: 587535

Country of ref document: AT

Kind code of ref document: T

Effective date: 20121215

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 602005037345

Country of ref document: DE

Effective date: 20130131

REG Reference to a national code

Ref country code: AT

Ref legal event code: MK05

Ref document number: 587535

Country of ref document: AT

Kind code of ref document: T

Effective date: 20121205

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20121205

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20121205

Ref country code: ES

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20130316

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20121205

REG Reference to a national code

Ref country code: NL

Ref legal event code: VDEP

Effective date: 20121205

REG Reference to a national code

Ref country code: LT

Ref legal event code: MG4D

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20121205

Ref country code: LV

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20121205

Ref country code: PL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20121205

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20130306

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: AT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20121205

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20121205

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20130405

Ref country code: CZ

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20121205

Ref country code: BG

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20130305

Ref country code: EE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20121205

Ref country code: SK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20121205

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20130405

Ref country code: RO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20121205

Ref country code: NL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20121205

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20121205

26N No opposition filed

Effective date: 20130906

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20121205

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20121205

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 602005037345

Country of ref document: DE

Effective date: 20130906

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20121205

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

REG Reference to a national code

Ref country code: IE

Ref legal event code: MM4A

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20131031

Ref country code: CH

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20131031

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20131020

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: TR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20121205

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: HU

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT; INVALID AB INITIO

Effective date: 20051020

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20131020

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 11

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 12

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 13

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 14

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: GB

Payment date: 20230831

Year of fee payment: 19

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: FR

Payment date: 20230911

Year of fee payment: 19

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: DE

Payment date: 20230822

Year of fee payment: 19